Recent decades have seen a dramatic increase in the incidence of a diverse range of fungal infections, including those of mucosal tissues and life threatening disseminated mycoses. The opportunist Candida albicans is a leading cause of both types of infection. The most widely used antifungal therapies act upon a narrow range of targets within the fungal cell, and have significant limitations including patient toxicity, limited formulations, and/or the development of resistance. The emergence of resistant C. albicans isolates, as well as intrinsically resistant species such as C. glabrata, is a serious medical concern. Thus there is an urgent need for new and improved treatment options. The goal of this proposal is to establish a new class of antifungal agents which act via a distinct mechanism from existing therapies. We will also begin to assess the potential of these agents as clinically applicable treatments. Our studies have demonstrated that disruption of the fungal vacuole renders C. albicans highly susceptible to host phagocytic cells, unable to invade mucosal tissue, and avirulent in a mouse model of disseminated infection. Similar studies have shown that the vacuole is essential for Cryptococcus neoformans to colonize and cause disease in a mouse model of cryptococcal meningoencephalitis. Therefore, the lack of an equivalent organelle in mammalian cells makes the vacuole an attractive intervention point to selectively disrupt fungal pathogenesis. We have devised an efficient high through-put method to identify small molecules which disrupt the integrity of the C. albicans vacuole. This will be used to screen a library of 50,000 'drug like' compounds. Preliminary data has established an expected 'hit rate' of 0.1-0.4%, thus we anticipate identifying 50-200 vacuole disrupting compounds. We will then progressively select those with the greatest potential for future clinical application. This will involve eliminating those which are toxic to mammalian cells, and confirming that each agent reduces the pathogenic potential of C. albicans. All fungi possess a vacuole; thus, in order to assess the 'broad spectrum' potential of each vacuole disrupting agent, we will test for activity against other infectious fungi including C. neoformans. The long-term goal will be to advance this new generation of antifungal agents towards clinical trials.